Efflux of beta-galactosidase products from Escherichia coli.

Several different strains of Escherichia coli were grown on a variety of carbon sources under various growth conditions. Lactose was added (usually at mid-log phase), and the concentrations of the products of beta-galactosidase action on this sugar (galactose, glucose, and allolactose) were determined at various times thereafter in the total culture and in the medium. It was found that with each strain, with all carbon sources, and under all of the conditions studied, a very large proportion of the products were found in the medium. Control studies were carried out which showed that these results were not artifacts of the method of separating the cells from the medium. The results also did not arise from the secretion of beta-galactosidase into the medium, from the diffusion of substrates and products into and out of the cells due to leaks in the membrane, or from faults in the method of sugar analysis. In addition, the results showed that there were very high levels of products inside the cells under the conditions used and that the efflux of the products was rapid. The efflux might be energetically advantageous to the cell as well as being a means of storing excess products until needed.     

Crystal Structure of the Periplasmic Component of a Tripartite Macrolide-Specific Efflux Pump

In Gram-negative bacteria, type I protein secretion systems and tripartite drug efflux pumps have a periplasmic membrane fusion protein (MFP) as an essential component. MFPs bridge the outer membrane factor and an inner membrane transporter, although the oligomeric state of MFPs remains unclear. The most characterized MFP AcrA connects the outer membrane factor TolC and the resistance-nodulation-division-type efflux transporter AcrB, which is a major multidrug efflux pump in Escherichia coli. MacA is the periplasmic MFP in the MacAB-TolC pump, where MacB was characterized as a macrolide-specific ATP-binding-cassette-type efflux transporter. Here, we report the crystal structure of E. coli MacA and the experimentally phased map of Actinobacillus actinomycetemcomitans MacA, which reveal a domain orientation of MacA different from that of AcrA. Notably, a hexameric assembly of MacA was found in both crystals, exhibiting a funnel-like structure with a central channel and a conical mouth. The hexameric MacA assembly was further confirmed by electron microscopy and functional studies in vitro and in vivo. The hexameric structure of MacA provides insight into the oligomeric state in the functional complex of the drug efflux pump and type I secretion system.     

Investigation on Stability of Transporter Protein, Glucuronide Transporter from Escherichia coli

The glucuronide transporter GusB, the product of the gusB gene from Escherichia coli, is responsible for detoxification of metabolites. In this study, we successfully expressed GusB homologously in E. coli and investigated its oligomeric state in n-dodecyl-β-d-maltoside (DDM) detergent solution. Evidence for a pentameric state with a Stokes radius of 57 ± 2 Å for the purified GusB protein in DDM solution was obtained by analytical size-exclusion HPLC. The elution peak corresponding to pentameric GusB is commonly seen in elution profiles in the different buffer systems examined over a wide pH range. Hence, it is likely that GusB resides in the membrane as a pentamer. Stability studies with different incubation periods with the typical lipids, such as dimyristoylphosphatidylcholine, and total E. coli phospholipids, as the representatives of both phosphatidylcholine and phosphatidylethanolamine, show some clues to two-dimensional crystallization of GusB with lipids.     

大肠杆菌的AcrAB-TolC多药外排泵及其调控研究进展

多药外排泵造成了细菌的多种药物的耐药现象, 这对感染性疾病的防治提出了挑战。对于多药外排泵的研究不仅使人们认识细菌耐药性机制, 而且为细菌耐药性的防治提供思路。大肠杆菌AcrAB-TolC外排泵系统的结构和调控机制研究取得了一些新进展, 这为病原菌的相关研究提供了参考, 本文对其进行了综述。     

Novel UGA-suppressors in Escherichia coli K-12

UGA-specific nonsense suppressors from Escherichia coli K-12 were isolated and characterized. One of them (Su+UGA-11) was identified as a mutant of the prfB gene for the peptide releasing factor RF2. It appears that in this strain, while peptide release at sites of UGA mutations is retarded, the UGA stop codon is read through even in the absence of a tRNA suppressor, exhibiting a novel type of passive nonsense suppression. Three suppressors (Su+UGA-12, -16 and -34) were capable of restoring the streptomycin sensitive phenotype in resistant bacteria (strAr). Because of their drug-related phenotype, these are possibly mutations in the components of the ribosomal machinery, particularly those concerned with peptide release at UGA nonsense codons. A tRNA suppressor was also obtained which was derived from the tRNA(Trp) gene. In this strain, a long region between rrnC (84.5 min) and rrnB (89.5 min) was duplicated and one of the duplicated genes of tRNA(Trp) was mutated to the suppressor. The mechanism of UGA-suppression is discussed in terms of translation termination at the nonsense codon in both active and passive fashions.     

Identification and Analysis of the Putative Pentose Sugar Efflux Transporters in Escherichia coli

Escherichia coli possesses a number of proteins that transport sugars out of the cell. We identified 31 candidate sugar efflux transporters based on their similarity to known sugar efflux transporters. We then tested whether these transporters affect arabinose and xylose metabolism. We identified 13 transporters - setC, cmr, ynfM, mdtD, yfcJ, yhhS, emrD, ydhC, ydeA, ybdA, ydeE, mhpT, and kgtP - that appeared to increase or decrease intracellular arabinose concentrations when respectively deleted or over-expressed. None of the candidate transporters affected xylose concentrations. These results indicate that E. coli possesses multiple arabinose efflux transporters. They also provide a novel target for future metabolic engineering.     

Novel compound for identifying Escherichia coli

A new chromogenic compound, 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronide, was found to be useful for the rapid, specific, differential identification of Escherichia coli in the sanitary analysis of shellfish and wastewater. Of 1,025 presumptively positive colonies (blue) and 583 presumptively negative colonies (nonblue), only 1% false-negative and 5% false-positive results were found.     

Efflux of choline and glycine betaine from osmoregulating cells of Escherichia coli

We present evidence that glycine betaine (betaine) which was synthesized from choline was excreted and reaccumulated in osmoregulating cells of Escherichia coli. Choline which was accumulated in bet mutants defective in betaine synthesis was shown to be excreted in response to betaine uptake. Our data suggest that E. coli has efflux systems for betaine and choline which are independent of the uptake systems for these metabolites. The ProU system of E. coli, but not that of Salmonella typhimurium, can mediate low-affinity choline uptake.     

The Multidrug Transporter LmrP Protein Mediates Selective Calcium Efflux

LmrP is a major facilitator superfamily multidrug transporter from Lactococcus lactis that mediates the efflux of cationic amphiphilic substrates from the cell in a proton-motive force-dependent fashion. Interestingly, motif searches and docking studies suggested the presence of a putative Ca(2+)-binding site close to the interface between the two halves of inward facing LmrP. Binding experiments with radioactive (45)Ca(2+) demonstrated the presence of a high affinity Ca(2+)-binding site in purified LmrP, with an apparent K(d) of 7.2 μm, which is selective for Ca(2+) and Ba(2+) but not for Mn(2+), Mg(2+), or Co(2+). Consistent with our structure model and analogous to crystal structures of EF hand Ca(2+)-binding proteins, two carboxylates (Asp-235 and Glu-327) were found to be critical for (45)Ca(2+) binding. Using (45)Ca(2+) and a fluorescent Ca(2+)-selective probe, calcium transport measurements in intact cells, inside-out membrane vesicles, and proteoliposomes containing functionally reconstituted purified protein provided strong evidence for active efflux of Ca(2+) by LmrP with an apparent K(t) of 8.6 μm via electrogenic exchange with three or more protons. These observations demonstrate for the first time that LmrP mediates selective calcium/proton antiport and raise interesting questions about the functional and physiological links between this reaction and that of multidrug transport.     

大肠杆菌的AcrAB-To1C多药外排泵及其调控研究进展

多药外排泵造成了细菌的多种药物的耐药现象,这对感染性疾病的防治提出了挑战.对于多药外排泵的研究不仅使人们认识细菌耐药性机制,而且为细菌耐药性的防治提供思路.大肠杆菌AcrAB-To1C外排泵系统的结构和调控机制研究取得了一些新进展,这为病原菌的相关研究提供了参考,本文对其进行了综述.     

Novel compound for identifying Escherichia coli.

A new chromogenic compound, 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronide, was found to be useful for the rapid, specific, differential identification of Escherichia coli in the sanitary analysis of shellfish and wastewater. Of 1,025 presumptively positive colonies (blue) and 583 presumptively negative colonies (nonblue), only 1% false-negative and 5% false-positive results were found.     

Reduction of Cellular Stress by TolC-Dependent Efflux Pumps in Escherichia coli Indicated by BaeSR and CpxARP Activation of spy in Efflux Mutants

Escherichia coli has nine inner membrane efflux pumps which complex with the outer membrane protein TolC and cognate membrane fusion proteins to form tripartite transperiplasmic pumps with diverse functions, including the expulsion of antibiotics. We recently observed that tolC mutants have elevated activities for three stress response regulators, MarA, SoxS, and Rob, and we suggested that TolC-dependent efflux is required to prevent the accumulation of stressful cellular metabolites. Here, we used spy::lacZ fusions to show that two systems for sensing/repairing extracytoplasmic stress, BaeRS and CpxARP, are activated in the absence of TolC-dependent efflux. In either tolC mutants or bacteria with mutations in the genes for four TolC-dependent efflux pumps, spy expression was increased 6- to 8-fold. spy encodes a periplasmic chaperone regulated by the BaeRS and CpxARP stress response systems. The overexpression of spy in tolC or multiple efflux pump mutants also depended on these systems. spy overexpression was not due to acetate, ethanol, or indole accumulation, since external acetate had only a minor effect on wild-type cells, ethanol had a large effect that was not CpxA dependent, and a tolC tnaA mutant which cannot accumulate internal indole overexpressed spy. We propose that, unless TolC-dependent pumps excrete certain metabolites, the metabolites accumulate and activate at least five different stress response systems.     

Substrate Selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli

Lysophospholipid transporter (LplT) was previously found to be primarily involved in 2-acyl lysophosphatidylethanolamine (lyso-PE) recycling in Gram-negative bacteria. This work identifies the potent role of LplT in maintaining membrane stability and integrity in the Escherichia coli envelope. Here we demonstrate the involvement of LplT in the recycling of three major bacterial phospholipids using a combination of an in vitro lysophospholipid binding assay using purified protein and transport assays with E. coli spheroplasts. Our results show that lyso-PE and lysophosphatidylglycerol, but not lysophosphatidylcholine, are taken up by LplT for reacylation by acyltransferase/acyl-acyl carrier protein synthetase on the inner leaflet of the membrane. We also found a novel cardiolipin hydrolysis reaction by phospholipase A₂ to form diacylated cardiolipin progressing to the completely deacylated headgroup. These two distinct cardiolipin derivatives were both translocated with comparable efficiency to generate triacylated cardiolipin by acyltransferase/acyl-acyl carrier protein synthetase, demonstrating the first evidence of cardiolipin remodeling in bacteria. These findings support that a fatty acid chain is not required for LplT transport. We found that LplT cannot transport lysophosphatidic acid, and its substrate binding was not inhibited by either orthophosphate or glycerol 3-phosphate, indicating that either a glycerol or ethanolamine headgroup is the chemical determinant for substrate recognition. Diacyl forms of PE, phosphatidylglycerol, or the tetra-acylated form of cardiolipin could not serve as a competitive inhibitor in vitro. Based on an evolutionary structural model, we propose a “sideways sliding” mechanism to explain how a conserved membrane-embedded α-helical interface excludes diacylphospholipids from the LplT binding site to facilitate efficient flipping of lysophospholipid across the cell membrane.     

Novel type of murein transglycosylase in Escherichia coli.

The purification and properties of a novel type of murein transglycosylase from Escherichia coli are described. The purified enzyme appears as a single band on sodium dodecyl sulfate-polyacrylamide gels and has an apparent molecular weight of approximately 65,000 as estimated by gel filtration and gel electrophoresis. It degrades pure murein sacculi from E. coli almost completely into low-molecular-weight products. The two prominent muropeptide fragments in the digest are the disaccharide-tripeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid and the corresponding disaccharide-tetrapeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid-D-alanine. The unique feature of these compounds is that the disaccharide has no reducing end group and that the muramic acid residue possesses an internal 1 leads to 6 anhydro linkage. The new lytic enzyme is designated as a murein: murein transglycosylase. Its possible role in the rearrangement of murein during cell growth and division is discussed.